Process for separation and collection of viable female and male spermatozoa

ABSTRACT

A process is disclosed for separating and collecting viable female spermatozoa (XX chromosome) and male spermatozoa (XY chromosome). The process utilizes apparatus comprising two sterilized columns of glass, plastic or other suitable material, a ball valve, a vacuum pump, a mercury manometer, and connecting tubes of glass, plastic or other suitable materials. The system is assembled with rubber percussion gaskets or other suitable connecting materials, to prevent the introduction of extraneous air into the closed system, and a burp bottle to preclude unwanted introduction of fluids into parts of the system under vacuum. The column containing the semen sample may be of variable volume, to accommodate semen samples of varying volume and concentration. 
     The process may be modified by introducing into the system a direct and continuous electrical current flow, or by creating within the separating columns an electrophoretic field. The process may be further modified into a continuous-flow system by adding another mercury manometer, a Cartesian Diver regulator, a second burp bottle, and an over-flow collecting bottle connected to the two main columns.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of co-pending U.S.application Ser. No. 805,869, filed June 13, 1977 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

A workable process for separating and collecting viable femalespermatozoa (XX chromosome) and male spermatozoa (XY chromosome) wouldbe of great value to the beef and dairy cattle industry by giving ahigher percentage male or female offspring, depending on demand. Theseparation of male and female components of semen samples would enhancethe probability of obtaining one sex over the other through anartificial insemination program by utilizing the proper semen fraction.

Accordingly, it is desirable to have a process for separating andcollecting viable female spermatozoa and male spermatozoa.

2. Description of the Prior Art

Sex differentiations between the male and female of mammalian speciesare very great. These differences are far too extensive to explain bysingle gene transmission. In most higher animals, there are two separatesexes and they have various degrees of differentiation of body parts.Involved in a great majority of forms of life that have separate sexesare the chromosomes. Since the XY method is the most common method ofsex determination, we will briefly say that within certain species thereare pairs of autosomes and one pair of X-chromosomes in the femalediploid cells, and pairs of autosomes and the paired X- andY-chromosomes in the male.

In oogenesis the chromosomes pair and the eggs and polar bodies allreceive the same kind of chromosomes--autosomes and one X-chromosome. Inspermatogenesis, one pair of chromosomes is unlike and half of the spermreceive the X-chromosome while the other half receives the Y-chromosome.Sex determination depends upon which of the two types of spermfertilizes the egg. Thus, we think of spermatozoa carrying theX-chromosome as being female-determining while those carrying theY-chromosome are male-determining. Segregation of the XY pair and randomfertilization explains superficially why some individuals develop intofemales and some into males. This is basically why in almost allmammalian species, about half of the members of each population aremales and half are females. Thus, the chromatin difference in thespermatozoa allows one to separate the X- from the Y- types of sperm,thereby being able to control the sex of the offspring at the time offertilization.

The determination of sex occurs at the time of fertilization dependingon whether the ovum is fertilized by an X- or Y-chromatin spermatozoa.The X-spermatozoa, uniting with the egg (ova) yields a female and theY-spermatozoa, a male. The female ovum is always the X-bearing chromatinmaterial. In most mammalian species the union of the two haploid germcells, the ovum X- with spermatozoa X- or Y-, the XX combination yieldsfemales and the XY combination yields males.

The introduction of artificial insemination as the preferential methodof insemination in cattle emphasized the importance of selecting bulldonors with regard to both their genetic quality and their fertilizingcapacity. Also, the longtime hope of sex control was revived because ofthe feasibility of treating semen prior to use in order to increase theincidence of desired sex.

The desire of man to be able to predetermine sex before conception andthus alter the natural sex ratio has prompted many investigations toseek methods of separating out two populations of sperm.

In the literature, no appreciable contributions have been discoveredtowards this problem. The following items cited in Table I are relatedto this effort.

                                      TABLE I                                     __________________________________________________________________________    Prior Art Citations                                                           No.                                                                              Citation                                                                   __________________________________________________________________________    1. ANNON. Aug. 22, 1949. Boy or Girl. Newsweek 34:44.                         2. BERNSTEIN, MARIANNE E. 1949. A son for every family? Science                  Digest 25:43.                                                              3. LUSH, J. L. 1925. The possibility of sex control by artificial                insemination with centrifuged spermatozoa. J. Agric. Research                 38:898-913.                                                                4. HARVEY, E. NEWTON. 1946. Can the sex of mammalian offspring be                controlled? J. of Heredity 37:71.                                          5. LINDAHL, PER ERIC. 1956. Counter-streaming centrifugation of bull             spermatozoa. Nature 178:491-492.                                           6. LINDAHL, PER ERIC, and KIHLSTROM, J. E. 1952. Alterations in                  specific gravity during the ripening of bull spermatozoa. J.                  Dairy Sci. 35:393-401.                                                     7. LINDAHL, PER ERIC. 1959. Separation of bull spermatozoa carrying              X- and Y- chromosomes by counter streaming centrifugation. Animal             Breeding Abstracts 27:758.                                                 8. LINDAHL, PER ERIC. 1952. On the relationship between fertility and            light-refracting power in bull spermatozoa. J. of Agric. Sci. 42.          9. ROBERTS, E. 1940. The effect of lactic acid and sodium bicarbonate            on the sex ratio. J. of Heredity 31:499-500.                               10.                                                                              WOLFF, ARTHUR. 1941. Sex control in mammals. Michigan St. Coll.               Vet. 1(2):53-54.                                                              COLE, LEON J., et al. 1940. A test of sex control by modification             of the acid-alkaline balance. J. of Heredity 31:501-502.                      COLE, L. J., et al. 1933. Sex control again. J. of Heredity                   24:265-274.                                                                   QUISENBERRY, J. H. 1945. Additional data on sex control in rabbits.           J. of Heredity 36:160.                                                        QUISENBERRY, J. H. and CHANDIRAMANI, S. V. 1940. An experimental              attempt to modify the sex ratio. J. of Heredity 31:503-505.                   McPHEE, H. C. and EATON, O. N. 1942. Experimental attempts to                 modify the sex ratio. J. of Heredity 33:429-433.                              CASIDA, L. E. and MURPHREE, R. L. 1942. Fertility and sex ratios              in the rabbit from semen treated in vitro with lactic acid and                sodium bicarbonate. J. of Heredity 33:434-438.                                WEIR, J. A. 1953. Association of blood pH with sex ratio in mice.             J. of Heredity 44:133-138.                                                    WEIR, J. A. 1955. Male influence on the sex ratio of offspring in             high and low blood pH. J. of Heredity 46:277-283.                             WEIR, J. A. 1953. Influence of the male on sex ratio of offspring             in high and low blood pH lines of mice. Genetics 38:700-701.               20.                                                                              PEARL, RAYMOND, et al. 1913. Data on sex determination in cattle.             Biol. Bull. 24:205-225.                                                       McWHIRTER, K. G. 1956. Control of sex ratio in mammals. Nature 178:           870-871.                                                                      SHETTLES, L. B. 1960. Nuclear morphology of human spermatozoa.                Nature 186:648-649.                                                           SHETTLES, L. B. 1960. Biology: X and Y spermatozoa. Nature 187:               254-255.                                                                      SHETTLES, L. B. 1960. Nuclear structure of human spermatozoa.                 Nature 188:918-919.                                                           SHETTLES, L. B. 1961. Human spermatozoa shape in relation to sex              ratios. Fertility and Sterility 12:502-508.                                   SHETTLES, L. B. 1961. Human spermatozoa types. Gynaecologia 162:              154-162.                                                                      SHETTLES, L. B. 1961. Sperm morphology and sex ratios. J. of                  Urology 86:450-455.                                                           SHETTLES, L. B. 1961. Conception and birth sex ratios. Obstet.                and Gynecol. 18:122-130.                                                      LUDWIG, WILHELM 1947. Uber Certation and spermiendimorphismus bei             tier und mnsch. Zeitscher. Naturforsch. 2b(5/6):222-226.                   30.                                                                              HARTMAN, CARL G. 1957. How do sperms get into the uterus? Fer-                tility and Sterility 8:403-427.                                               R-PHYSIOLOGISTS. Personal communication.                                      DUIJN, C. VAN 1960. Nuclear structure of human spermatozoa.                   Nature 188:916-918.                                                           ROTHSCHILD, LORD. 1960. Biology: X and Y spermatozoa. Nature                  253-254.                                                                      SHRODER, VERY N. 1933. Artificial control of sex in the progeny of            mammals. Nature 131:329.                                                      SHRODER, VERA N. 1932. Die physikalisch-chemische analyse einiger             fragen der spermien-physiologie. Biol. Zhur. 1:24-29.                         SHRODER, VERA N. 1934. Uber ionequilibrierte verdunnungslosungen              fur die pferdespermien. Biol. Zhur. 3:465-476.                                SHRODER, VERA N. 1941. Kunstliche geschlects-regulation der nach-             kommenschaft der saugetiere und ihre biologishe kontrolle. Zeitschr.          fur Tierzuchtung und Zuchtungsbiol. 50:1-15.                                  SHRODER, VERA N. 1941. Uber die biochemischen und physiologischen             eigentumlichkeiten der X- and Y- spermien. Zeitschr. fur Terizuch-            tung und Zuchtungsbiol. 50:16-23.                                             MACHOWKA, W. W. and SCHEGALOFF, S. B. 1935. Die reaktion der                  spermatozoen auf konstanten strom (galvanotaxis). Archiv. fur                 Entwicklungsmechanik der Organismen. 133:694-700.                          40.                                                                              KORDTS, E. 1952. Untersuchungen uber die eignug der elektrophorese            zur trennung der mannchen und weilchenbestimmenden spermien biem              kaninchen. Zeitschr. fur Tierzuchtung und zuchtungsbiol. 60:221-240.          PILZ, A. 1952. Das verhalten der saugetiersspermien in elektrischen           feld. Zietschr. fur Tierzuchtung und Zuchtungsbiol. 60:315-330.               GORDON, MANUEL J. 1957. Control of sex ratio in rabbits by electro-           phoresis of spermatozoa. Proc. Natnl. Acad. Sci. 95:913-918.                  GORDON, MANUEL J. 1958. The control of sex. Sci. Amer. 199:87-94.             LEWIN, SHERRY 1956. Artificial sex regulation of mammalian off-               spring. Brit. Vet. J. 112:549-550.                                            MacPHERSON, J. W. and VESSELINOVITCH, S. D. 1959. Electrophoresis             of bovine semen. Can. J. of Comp. Med. and Vet Sci. 23:375-376.               VESSELINOVITCH, S. D. and MacPHERSON, J. W. 1959. Electrophoresis             of bovine spermatozoa. The Cornell Vet. 49:359-373.                           VESSELINOVITCH, S. D. 1959. Microelectrophoresis of bovine sperma-            tozoa. Can. J. of Comp. Med. and Vet. Sci. 23:1-19.                           VESSELINOVITCH, S. D. 1960. Electrophoresis of spermatozoa and sex            control. Cornell Vet. 50:326-330.                                             DIASIO, R. and GLASS, R. 1971. Effects of pH on the migration of              X and Y sperm. Fertility and Sterility 22:5.                               50.                                                                              EMMERICH, E. and STOLKOWSKI, J. 1970. Influence of Mineral nutri-             tion on sex distribution in the cow: prospective and experimental             investigations. Enn. Endoc. (Paris) T. 31:2.                                  MORGAN, D. and ROAN, C. 1972. Nature 238:233.                                 SEVINC, A. 1968. Experiments on sex control by electrophoretic                separation of spermatozoa in the rabbit. J. Reprod. Fer.                   __________________________________________________________________________       16:7-14.                                                               

There are records of Greek physicians in 500 B.C. advising theirpatients, expectant mothers, to lie on their right side while sleepingand they would surely bear a son. If they slept on their left sides,they will give birth of a daughter (see Citation No. 1 in Table I).

It was not until after the Nobel Prize winning discovery of thesex-determining X- and Y-chromosomes by Professor T. H. Morgan in 1912(see Citation No. 2 in Table I) that any scientifically based theorieswere propounded on the control of the sex of the offspring. The majorityof these theories are founded on the concept that the main differencesbetween the spermatozoa which carry the larger X-chromosome (femaleproducing) and the spermatozoa which carry the smaller Y-chromosome(male producing) are: weight, size, speed of locomotion, viability atvarious pH values, and over-all electric charge.

In 1925, Lush (see Citation No. 3 in Table I) made many attempts tocontrol the sex in rabbits by separating spermatozoa according to sizeby partial centrifugation and inseminating the fractions in females. Hebelieved that there was dimorphism between the two types of spermatozoa,but stated that the observable difference as seen through the microscopewas very small. The results of his work show that there was nosignificant deviation from the expected ratio of males to females in theoffspring.

Harvey (see Citation No. 4 in Table I) in 1946 modified Lush'sprocedure. He believed that the size of the spermatozoa were all thesame but that the X-chromosome-carrying spermatozoa would be more densethan the Y-chromosome-carrying spermatozoa due to the mass difference ofthe two chromosomes. If this were true, then a solution could beprepared which would intermediate in density between the two types ofspermatozoa at a given temperature. By centrifugation the less dense Yspermatozoa would float while the more dense X spermatozoa would sink.Although Harvey did not test his theory by insemination, he calculatedthat the difference in the two types of spermatozoa is of the order of 2in 10,000, a separation comparable to the separation of Uranium 235 fromUranium 238.

Lindahl (see Citation No. 5 in Table I) in 1956 applied the theory ofseparation of spermatozoa to cattle using the counter-streamingcentrifuge. In this process, he collected the heavier spermotozoa(theoretically those with the X-chromosome) and inseminated them. Thisdid not result in any significant divergence from the normal sex ratio.He cited Lindahl and Kihlstrom (see Citation No. 6 in Table I) asstating " . . . the density of bull spermatozoa increases in the courseof their physiological maturation. This change in density is of such anorder of magnitude that it totally obscures the difference insedimentation rate due possibly to the difference in volume between thesex chromosomes." It could be possible that, in general, the X-carryingspermatozoa are usually larger and heavier than the Y-carryingspermatozoa, if there is no consistent amount of cytoplasm retained bythe spermatozoa in maturation, or if there is no change in size as theage of the spermatozoon increases. If the amount of cytoplasm retainedby the spermatozoon is inconsistent, then the relative differences inthe masses of the sex chromosomes might not be sufficient to render thetwo types of spermatozoa separable by these methods. The aboveexperiments, within their limits, seem to say that there is nocorrelation between the weight, size, or density of the spermatozoa andthe type of sex chromosome it carries.

In 1959 Lindahl (see Citation No. 7 in Table I), using the counterstreaming centrifugation method, produced a "heavy" and "light" sperm.The "heavy" and the "light" spermatozoa were used for the inseminationof 142 and 121 cows, respectively. The difference between the two groupsas regards fertility and sex ration was not statistically significant,but, within each group, higher fertility was significantly associatedwith a greater proportion of female calves born and, in the first group,with a lower centrifuge speed. Counter-streaming centrifugation of bullsemen was carried out at velocities of 100-200 rpm. The results indicatethat female determining spermatozoa are more liable to damage duringcentrifugation than are male determining spermatozoa. Fertilitydecreased more at the higher than at the lower velocities.

Lindahl (see Citation No. 8 in Table I) in 1952 also reports that thespecific gravity of bull spermatozoa increases during ripening of thecells. This process is most pronounced at maturation due to loss ofresidual protoplasm, but continues also later and seems to form part ofthe changes underlying "over-ripening". The rise in density probablydepends upon loss of water accompanied by a corresponding decrease involume. However, the water still present is extremely firmly bound andresists high osmotic pressures (186 atmospheres). The ripe spermatozoainclude a series of ripening stages, one of which represents the maximalfertile state.

In 1940 Roberts (see Citation No. 9 in Table I) douched the vagina offemale rats with weak solution of lactic acid or sodium bicarbonate lessthan two hours before mating. In the cases in which he used lactic acidhe found that in 103 litters there were 280 males and 467 females. Whenhe used sodium bicarbonate he found that in 104 litters there were 420males and only 200 females. This would indicate that variation ofvaginal pH could influence the ratio of the sexes of the offspring.Roberts offered no theories to explain his results, but it would appearthat the pH in the vagina affected either the survival time of one ofthe two types of spermatozoa or affected the fertility of one type ofspermatozoa.

Wolff (see Citation No. 10 in Table I) found in 1941 that the pH of thevagina at fertilization altered the normal sex ratio of the offspring.He reported that bicarbonate ions would produce more male while lacticacid would produce more female offspring, which is in agreement withRoberts (see Citation No. 9 in Table I).

At this time Cole, et al. (see Citation Nos. 11 and 12 in Table I) wereperforming this experiment with both rats and rabbits. Using lactic aciddouches they found that in rats 447 males were produced to 408 females.When sodium bicarbonate was used 452 males and 439 females resulted,almost a 1:1 sex ratio in both instances. Fewer rabbits were obtained,but their sex ratio was almost 1:1 in each case. They went a stepfurther by inseminating female rabbits with semen treated with either aweak acid or base, but only negative results were obtained.

Quisenberry (see Citation No. 13 in Table I) and Quisenberry andChandiramani (see Citation No. 14 in Table I) were working on thisproblem in 1940 and found no significant divergence from the expectedsex ratio when they used the vaginal douche technique in rats andrabbits.

McPhee and Eaton (see Citation No. 15 in Table I) working with rabbitsand swine in 1942 failed to find any significant modification of thenormal sex ratio as a result of acid or alkali douche treatments in2,383 rabbit offspring and 219 swine offspring. Casida and Murphree (seeCitation No. 16 in Table I) in 1942 found no change in the expected sexratio of the offspring of rabbits when vaginal douches of various pHvalue were administered prior to fertilization.

Although Roberts and Wolff (see Citations No. 9 and 10, respectively, inTable I) did meet with success in their work with vaginal douches in thecontrol of sex of the offspring, many others did not.

Weir (see Citations 17, 18 and 19 in Table I) began in 1953 a new seriesof experiments in which he developed two strains of mice, one with anaverage blood pH of 7.42 and the other with an average blood pH of 7.46.He found that the interbreeding of those mice with the blood pH of 7.42altered the female/male sex ratio from 50:50 to 60:40 and theinterbreeding of the mice with the blood pH of 7.46 altered the ratio toabout 40:60. By cross breeding or reciprocal mating of different sexesof the two strains, he demonstrated that it was the blood pH of the maleand not the female which determined the sex ratio of the young. Weirtheorized that " . . . The association of an excess of females with lowblood pH suggests that the genetic constitution of the mother may set upchemical conditions which in turn may act on the spermatozoa of thefather to favor or handicap either the X or Y type, making fordifferential sex ratios in the resulting litters."

McWhirter (see Citation No. 21 in Table I) in 1956 tried to determinewhether Weir's idea could be applied to other mammals and whether theblood pH changes which occur or which could be developed by selectivebreeding could affect the sex ratio of the offspring. He came to believethat significant variation of the sex ratio could be accomplished inother animals if a selected sire with the appropriate blood pH was usedto father the offspring. The major objection to this method is that onlycertain males could be used if a particular sex offspring were desired.

Pearl, et al. (see Citation No. 20 in Table I) proposed in 1913 a methodin which the time of copulation or insemination was varied with respectto the time of ovulation or period of "heat". They observed that whencopulation in cattle took place during early heat, 31 males and 51females were conceived and born. When copulation took place in lateheat, 42 males and only 34 females were produced. They summarized: "Asthe time of coitus approaches the end of the oestrous period, there is aprogressive increase in the proportion of male young born."

Shettles (see Citations 22 through 28 in Table I) in 1960 and 1961 hasmade some startling discoveries which seem to support this theory. Usinga phase contrast microscope for observation of human spermatozoa hefound two distinct populations of spermatozoa with regard to head andnuclear size and shape. One population was larger and had ovoid headswith a nucleus of similar shape. These were fewer in number than theother population which was smaller and had a round head containing around nucleus. He theorized the larger spermatozoa were those containingthe larger X-chromosome and would therefore produce female progeny whilethe smaller spermatozoa were those containing the X-chromosome and wouldproduce male offspring. He went a step further in an attempt to explainthe time factor of fertilization and its effect on the sex ratio. Hetheorized that since the Y-spermatozoa were smaller than theX-spermatozoa, they could swim faster. If insemination were immediatelybefore ovulation, the Y-spermatozoa would reach the egg first and theresulting zygote would be male. (Ludwig (see Citation No. 29 in Table I)agrees with this theory.) If insemination were some time prior toovulation, then the weaker Y-spermatozoa would not be able to survivethe conditions in the female reproductive tract as well as theX-spermatozoa and would be fewer in number. Therefore, at fertilizationthe probability that the zygote would be female would be much higher.

According to Hartman (see Citation No. 30 in Table I) the major role inthe migration of spermatozoa in humans to the site of fertilization isplayed by muscular contraction of the walls of the female reproductivetract. He states that sexual stimulation through neural pathways causesan output of oxytocin which increases the muscular activity of thegenital tract of the female. He further states that the main function ofthe tail of the spermatozoa is to effect the penetration of the coronaradiata cells of the ovum itself. Some reproduction physiologists (SeeCitation No. 31 in Table I) are in agreement that the swimming of thesperm in the female reproductive tract could be compared to a man tryingto swim in a hurricane. The massive movements of the ocean (fluid inuterus and oviducts) would be much greater than the feeble swimming ofman (spermatozoa). If one holds to the latter beliefs, then he could notsay that the Y-spermatozoa swim faster and reach the egg first.

Other points of conflict with Shettles' work have been raised by C. vanDuijn and Rothschild (see Citation Nos. 32 and 33, respectively, inTable I). Rothschild states that there is so far no evidence thatphysical differences have been found between the X- and Y-spermatozoa,using phase contrast microscopy. C. van Duijn believes that Shettles isseeing artifacts due to a maladjusted optical element in his phasecontrast microscope.

Vera N. Shroder (sometimes spelled Shreder) (see Citation Nos. 34 and 35in Table I) in 1932 studied the behavior of cells in an electric field.She found that most cells will migrate, in electrophoresis, to the anodeor positive pole. When she introduced rabbit spermatozoa, suspended inphysiological solution at pH 7.1, into a Michaelis or Kross-Zuelzerapparatus, she observed that about one-half of the cells migrated to theanode and the other half migrated to the cathode when an electricalcurrent was applied. Shroder then inseminated three female rabbits withthree electrophoretically separated portions of spermatozoa. The oneimpregnated with the spermatozoa which migrated to the anode bore 6young, all of them females. The one impregnated with the spermatozoafrom the cathode bore 5 offspring, 4 males and 1 female. With thesuspension of spermatozoa remaining in the middle between the twoelectrodes, she impregnated another female rabbit. Of the 4 young thusproduced, 2 were males and 2 were females. She theorized that theseresults were due to the separation of the spermatozoa in theelectrophoretic apparatus into two populations--one populationconsisting predominately of Y-bearing spermatozoa and the other ofX-bearing spermatozoa. She believed that if the pH of the physiologicalsolution were intermediate between the isoelectric points of the twopopulations of spermatozoa, then the two populations would exhibitopposite charges. This would appear to be verified by her observationsand experiments. Shroder developed the technique and procedure to thepoint that it was possible to predict the sex of the offspring ofrabbits with 80% accuracy (see Citation Nos. 36, 37 and 38 in Table I).

In 1935 Machowka and Schegaloff (see Citation No. 39 in Table I)attempted to duplicate Shroder's work. They observed the two-waymigration of spermatozoa and inseminated female rabbits with fractionsobtained by electrophoresis in the Michaelis apparatus. In 50 litters,216 young were produced. Of these, 105 were females and 111 were males,almost the ratio which would be expected under normal breedingconditions. As a result of their work, they rejected Shroder's theory ofdifferent isoelectric points for each of the two types of sperm. Theytheorized that all mammalian cells possess a negative charge, and thatonly in adverse conditions with the lipid component of the cell membranebe destroyed and positive ions become absorbed to the cell to such anextent that the over-all, net charge of the cell will become positive.They therefore speculated that the two-way migration of the spermatozoais due to two things. One is a passive cataphoretic movement and theother is an active negative galvanotaxis. They did not believe that thetwo-way migration is due to the difference in the X- and Y-chromosomes.

Kordts (see Citation No. 40 in Table I) in his experiments in 1952 onelectrophoresis of rabbit spermatozoa observed a marked separation ofspermatozoa. Upon subsequent insemination of the fractions in females,he obtained 127 offspring, the sex ratio being 48.4% males to 51.2%females.

Pilz (see Citation No. 41 in Table I) was working with both rabbit andbull spermatozoa. He did not have success in the two-way separation ofthe spermatozoa. He did observe, however, that inactive and/or deadspermatozoa may migrate to either the cathode or anode. Since heobtained no impressive separations, he did not attempt anyinseminations.

Gordon (see Citation Nos. 42 and 43 in Table I) appears to havesuccessfully separated rabbit spermatozoa by means of electrophoresisinto fractions. From 201 inseminations, he obtained 31 litters and atotal of 167 offspring. His accuracy in the prediction of the sex of theoffspring was 71.3% for females and 63.7% for males. In the apparatus heused for electrophoresis, only one pole was accessible for extraction ofspermatozoa. Therefore, it was necessary to make two trials, one withthe poles reversed, in order to get the two samples of spermatozoa fromboth the positive and negative poles.

Gordon theorized that the migrations were due to sexual dimorphism inthe spermatozoa which was the result of a difference in their surfacecharges. He further stated that there was no proof for electrophoreticseparation due to surface charges, as Shroder observed that thetemperature would affect the direction of migration. Gordon interpretedthis as follows: "At higher temperatures, the surface membranes,normally acting as electrical insulators, may lose this property andthen act as conductors, so that internal particles having charges may beresponsible for migration." The difference in the internal charge wouldbe due to the differences in the X- and Y-chromosomes.

Lewin (see Citation No. 44 in Table I) observed two-directionalmigration of spermatozoa in an electric field in both rabbit and man.She stated that the ova of several species can be shown to migrate in anelectric field and that a charged ovum could be selectively fertilizedby an oppositely charged spermatozoon as particles with like chargesrepel and particles with unlike charges attract. The charges exhibitedby the ovum and spermatozoon are a function of the pH, therefore, the pHof the medium in which fertilization takes place could favor one sexover the other. She went a step further to state that if one were tocombine electrophoresis with Weir's principle (see Citation Nos. 17, 18and 19 in Table I) then it might be possible to predict the sex of theprogeny with greater accuracy than 80%.

Vesselinovitch and MacPherson (see Citation Nos. 45 and 46,respectively, in Table I) and Vesselinovitch (see Citation Nos. 47 and48 in Table I) have done much work on the electrophoresis of bullspermatozoa. They reported that in no case did they note anytwo-directional movement of spermatozoa due to electrophoresis. They didobserve that first, the immotile spermatozoa were carried to the anodeby electrophoresis; second, that moderately active spermatozoa swimactively, after undergoing galvanotaxis, to the cathode; and third, thatthe highly active spermatozoa swim actively at random. In subsequentinseminations of anode- and cathode-spermatozoa, they observed nosignificant divergence from the normal sex ratio of the offspring. In1960 Vesselinovitch (see Citation No. 48 in Table I) states: "Inconclusion it may be said that on the basis of the previously reportedwork and comments made here, we believed that it is sound to state thatthere are no solid grounds at present on which to assume that trueelectrophoresis of spermatozoa may solve the problem of sex control."

Observations on Nuclear Morphology of normal human sperm by Dr. LandrumB. Shettles of Columbia-Presbyterian Medical Center in New York City(1961) finds that there are two populations of cytologically normalsperm in regard to head and nuclear size and shape: larger, oval, andsmaller, rounded types. There were no intermediate types. The smallerand rounded heads contained a centrally located chromosome, whereas thelarger ones had a centrally located elongated chromosome.

Robert B. Diasio, et al., (see Citation No. 49 in Table I), studying theeffects of pH upon sperm migration found that pH did not affect sperm incapillary tubes. They state that on both clinical and experimentalgrounds it appears unlikely that the X and Y sperm can be differentiatedon the basis of migration through fluids of varying pH's.

The following U.S. patents and literature reference are also cited toexemplify the state of the prior art:

U.S. Pat. Nos. 3,687,806--Aug. 29, 1972--Van Den Bovenkamp;3,873,432--Mar. 25, 1975--Israel, et al.; 3,894,529--July 15,1975--Shrimpton; 3,906,929--Sept. 23, 1975--Augspurger; 3,914,168--Oct.21, 1975--Allington; 3,976,197--Aug. 24, 1976--Bhattacharya;4,007,087--Feb. 8, 1977--Ericsson; 4,009,260--Feb. 22, 1977--Ericsson.

Smith "Chromatographic and Electrophoretic Techniques" (1960), pages120, 121, 123, 137.

SUMMARY OF THE INVENTION

This invention relates to a process for separating and collecting viablefemale spermatozoa (XX chromosome) and male spermatozoa (XY chromosome).The process comprises two sterilized columns of glass, plastic or othersuitable material, joined together by a ball valve, and rubberpercussion gaskets or other suitable connecting materials, a vacuumpump, a mercury manometer, and connecting tubes of glass, plastic orother suitable materials. The system is assembled so as to prevent theintroduction of extraneous air into the closed system. The columncontaining the semen sample may be of variable volume, to accommodatesemen samples of varying volume and concentration.

The process may be modified by introducing into the system a direct andcontinuous electrical current flow, or by creating within the separatingcolumns an electrophoretic and/or electrostatic field. The process maybe further modified into a continuous-flow system by adding anothermercury manometer, a Cartesian Diver regulator, burp bottle, and anover-flow collecting bottle connected to the two main columns.

Accordingly, a primary object of this invention is to provide a processfor separating and collecting viable female and male spermatozoa.

Another object of the invention is to provide a process for suchseparation and collection wherein such spermatozoa are subjected to adeficiency or excess of oxygen.

Yet another object of the invention is to effect this separation andcollection process by using manometric pressure in conjunction with adirect and continuous electrical current flow or an electrophoreticand/or electrostatic field.

Yet a further object is to effect the process with negative manometricpressure.

A further object of the invention is to provide a semen sample column ofvariable volume, to accommodate semen samples of varying volume andconcentration.

A still further object of the invention is to provide a continuous-flowprocess for separating and collecting viable female and male spermatozoaby introducing an additional manometer, a Cartesian Diver regulator,burp bottle, and over-flow collecting bottle.

These together with other objects and advantages which will becomesubsequently apparent reside in the details of construction andoperation as more fully hereinafter described and claimed, referencebeing had to the accompanying drawings forming a part hereof, whereinlike numerals refer to like parts throughout.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a side elevational view of the apparatus designed to separateand collect viable female spermatozoa (XX chromosome) and malespermatozoa (XY chromosome) by subjecting the sperm sample to manometricpressure.

FIG. 2 is a side elevational view of the apparatus of FIG. 1, with theaddition of a method to subject the sperm sample to a direct andcontinuous electrical current flow.

FIG. 3 is a side elevational view of the apparatus of FIG. 1, with theaddition of a method to subject the sperm sample to an electrophoreticand/or electrostatic field.

FIG. 4 is a side elevational view of the apparatus of FIG. 3, with theaddition of a method to make a continuous-flow system by introducinginto the system an additional mercury manometer, a Cartesian Diverregulator, burp bottle, and an over-flow collecting bottle attached tothe two main columns.

FIG. 5 is a side elevational view of the apparatus of FIG. 2, with theaddition of a method to make a continuous-flow system by introducinginto the system an additional mercury manometer, a Cartesian Diverregulator, burp bottle, and an over-flow collecting bottle attached tothe two main columns.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In FIG. 1 of the drawings, there is disclosed apparatus useful in aprocess for separating and collecting viable female spermatozoa (XXchromosome) and male spermatozoa (XY chromosome). Semen sample 10 isintroduced into lower chamber 12, according to semen concentration andvolume. The volume adapters 14, 16, 18 and 20 are designed to holdvolumes of 30, 50, 75 and 100 milliliters, respectively, and can be madeto accommodate any volume. The diameter of these adapters is 3/4 inch,but may be varied.

Upper chamber 22 of the column will preferably be longer than theadapter portion, in lower chamber 12, to allow for turbulence andburping, and so that an extender may be introduced in an equal volume tothe semen sample in lower chamber 12.

Valve 24 of the column is a 3/4 inch ball valve, thus allowing closurebetween lower chamber 12 and upper chamber 22.

Container 26 is a burp bottle to eliminate the possibility of fluidsbeing drawn into vacuum pump 28, which is conventional in construction.Container 26 can be vented by valve 30 through vent tube 32 to theatmosphere. In another position of valve 30, line 34 is open tocontainer 26, or in a third position of valve 30, both line 34 and tube32 are closed to container 26. Manometer tube 36 and scale 38 are usedto monitor the pressure during the process by measurement of the heightof mercury drawn from reservoir 40 as pump 28 evacuates container 26 andtube 36 through line 42.

The entire apparatus constitutes a closed system held together by rubberpercussion gaskets 44 or other suitable means, to prevent theintroduction of extraneous air.

The general procedure for operation is as follows: a semen sample isintroduced into lower chamber 12, valve 24 is then closed, and theextender is introduced into upper chamber 22. Chambers 12 and 22 containliquid of equal volume. The column is then subjected to a manometricpressure of twelve inches of mercury, and valve 24 is gently opened. Thesystem is subjected to this pressure until column turbulence ceases.Negative pressure is then increased to the maximum, approximately thirtyinches, for about one hour, with variations in maximum pressure and timelength selected according to the physiology of the semen, andatmospheric conditions.

The male spermatozoa (XY chromosome) will react to these pressuresbefore the female spermatozoa (XX chromosome), and will accumulate atthe top of the columns.

In FIG. 2 the sperm separation process is identical to the processdescribed in FIG. 1, except that a varying direct and continuouselectrical current flow of approximately six volts is generated by powersupply 49 and introduced by electrode rods 50 and 52 held within lowerchamber 54 and upper chamber 56, respectively, through the entire lengthof the separating column.

The semen sample is introduced into lower chamber 54, according to semenconcentration and volume. Volume adapters 58, 60, 62 and 64 are designedto hold volumes of 30, 50, 75 and 100 milliliters, respectively, and canbe made to accommodate any volume. The diameter of these adapters is 3/4inch, but may be varied.

Upper chamber 56 of the column will always be longer than the adapterportion, to allow for turbulence and burping, and so that the extendermay be introduced in an equal volume to the semen sample in lowerchamber 54.

Electrode rods 50 and 52 in chambers 54 and 56, preferably stainlesssteel or other suitable electrically conductive material, are sealedcoaxially into the chambers to prevent introduction of extraneous air orleakage of fluids, and are held in contact with valve 60 by electricallyconductive springs 62 and 64.

Valve 60 of the column contains a 3/4 inch ball valve of suitableelectrically conductive material, thus allowing closure between lowerchamber 54 and upper chamber 56.

Connecting wire 66 of suitable electrically conductive material permitsthe entire separating column to be subjected to a direct and continuouselectrical current flow.

As in FIG. 1, container 26 in FIG. 2 is a burp bottle to eliminate thepossibility of fluids being drawn into the vacuum pump and manometertube 36, scale 38, and reservoir 40 are used to monitor the pressureduring the process.

Lamp 68 indicates when direct and continuous electrical current flows.

The column is a closed system held together by rubber percussion gaskets70 or other suitable means, to prevent the introduction of extraneousair.

The operation of the system described in FIG. 2 is identical to theoperation of the system described in FIG. 1.

In FIG. 3 of the drawings, the sperm separation process is identical tothe process described in FIG. 1, except that an electrophoretic and/orelectrostatic field has been introduced in the separating column.

The semen sample is introduced into lower chamber 72, according to semenconcentration and volume. The volume adapters 74, 76, 78 and 72 aredesigned to hold volumes of 30, 50, 75 and 100 milliliters,respectively, and can be made to accommodate any volume. The diameter ofthese adapters is 3/4 inch, but may be varied.

Upper chamber 80 of the column will always be longer than the loweradapter portion, to allow for turbulence and burping, and so that theextender may be introduced in an equal volume to the semen sample inlower chamber 72.

Valve 82 is a 3/4 inch ball valve, thus allowing closure between upperchamber 80 and lower chamber 72.

Container 26 and associated components function as described above forFIG. 1 to eliminate the possibility of fluids being drawn into vacuumpump 28, and components to monitor the pressure during the process areas described above for FIG. 1.

Direct current is supplied by power supply 49 for the electrophoreticand/or electrostatic field. The intensity of the field is controlled bydirect current power supply 49 and the distance between the two poles,and will vary according to the physiology of the semen sample.

The operation of the system described in FIG. 3 is identical to theoperation of the system described in FIG. 1, except that the electricalcurrent is applied and the electrophoretic and/or electrostatic field iscreated after maximum negative pressure is reached.

In FIG. 4 of the drawings, there is disclosed a continuous-flow processfor separating and collecting viable female spermatozoa (XX chromosome)and male spermatozoa (XY chromosome).

Lower chamber 84 and upper chamber 86 are first filled with extender tothe level of over-flow collecting bottle 88.

The concentrated semen sample is introduced into reservoir 90, withstop-cock 92 at the bottom of reservoir 90 in a closed position.

Valve 94 is a 3/4 inch ball valve, thus allowing closure between upperchamber 86 and lower chamber 84.

Volume adapters 96, 98 and 100 of 30, 50 and 75 milliliters,respectively, can be made to accommodate any volume and can besubstituted for lower chamber 84.

The continuous system operates on a variable pressure. Semen reservoir90 is subjected to two inches less mercury pressure than chambers 84 and86, valve 94, and over-flow collecting bottle 88. This is accomplishedby the Cartesian Diver regulator, designated by the numeral 102 in FIG.4. Mercury manometer tubes 104 and 106 monitor pressures during theprocess.

Containers 108 and 110 are burp bottles to eliminate the possibility offluids being drawn into vacuum pump 28 or into either regulator 102 orreservoir 90.

Direct current is supplied by power supply 112 for the electrophoreticand/or electrostatic field. The intensity of the field is controlled bydirect current power supply 112, and will vary according to thephysiology of the semen sample.

To operate the continuous flow system, the concentrated semen sample isfirst introduced into reservoir 90. Reduced pressures are introduced at10 inches mercury on manometer tube 106, and 8 inches mercury onmanometer tube 104, for approximately 15 minutes, or until turbulenceceases. Valve 94 is then opened and after turbulence has again subsided,pressure is decreased to approximately 30 inches of mercury on manometertube 106 and 28 inches of mercury on manometer tube 104. Valve 94 isopen during this process. After turbulence has subsided, stop-cock 92 atreservoir 90 is slowly opened, allowing the total volume of the semensample to proceed through the separating column from reservoir 90 toover-flow collecting bottle 88 in one hour. Valve 94 is then closed. Thesemen in over-flow collecting bottle 88 contains predominantly maleproducing spermatozoa (XY chromosome), and lower chamber 84 containspredominantly female producing spermatozoa (XX chromosome).

In FIG. 5 of the drawings, the sperm separation process is identical tothe process described in FIG. 4, except that in FIG. 5 connecting wire114 of suitable electrically conductive material permits the entireseparating column to be subjected to a direct and continuous electricalcurrent flow, generated by direct current power supply 112.

The operation of the system described in FIG. 5 is otherwise identicalto the operation of the system described in FIG. 4.

Since the present invention can be practiced in the manner taughtherein, without regard to the explanation of the theory and principlesresponsible for the effects taught, the explanations advanced herein areintended to in no way limit the scope of the present invention definedby the claims.

Cellular respiration may be defined as the osmotic and chemical processor processes by which a plant or animal adsorbs oxygen and gives off theproducts formed by the oxidation in the tissues.

Spermatozoa carry on cellular respiration and our work demonstrates thatthere are substantial differences in the amounts of oxygen consumedbetween the X- and Y-spermatozoa populations. Oxygen consumption isrelated to the separation of the X- and Y-spermatozoa due to theirrespiration.

The present invention apparatus is a closed system and oxygen is removedfrom the top of the system. Semen samples are placed in the bottomportion of the system, with the same amount of extender placed in thetop portion of the system, and the removal of oxygen from the systemcauses the spermatozoa that respire faster to migrate to the upperportion of the column. Since one population consumes more oxygen thanthe other population of spermatozoa, the oxygen deprived spermatozoathat respire at a faster rate, migrate to the top of the column.

By definition, a semen sample contains spermatozoa of the X- andY-types, glandular fluids and other reproductive organ fluids from themale of the species. The extender is any commercial extender used inartificial insemination work of that particular species.

X- and Y-mammalian spermatozoa have not been separated, to any degree,due to their size variations. Also, they have not been separated to anydegree by different weights of the two. This is due to theirinfinitesimally small differences both in weight and size. Thesevariations cannot be detected under the (ordinary) light microscope.However, these variations can be detected by the use of the electronmicroscope, as well as phase contrast miscroscopy. We have done thiswith the electron microscope and the literature bears out these sizevariations in the chromatin mass of the X- and Y-mammalian spermatozoa.

By changing the osmotic pressures within the closed system, aspreviously mentioned, it is possible to utilize the difference in sizeand weight of the X- and Y-spermatozoa in the separation procedure.Utilizing this force the present invention has enhanced the separationof the X- and Y-spermatozoa as shown in the apparatus in FIG. 1.

The electro-potential energy differences between the X- and Y-mammalianspermatozoa are explained by relating them to the maturation process ofthe animal germ cell.

In spermatogenesis, the X- and Y-spermatids are formed during meiosis(second division of metaphase on the chart), a special division of theanimal germ cells. Somatic cell division within the body takes place dueto a phenomenon known as mitosis. Germ cell division takes place similarto mitosis, but an additional stage called meiosis allows for theproduction and maturation of the sperm and egg to be developed. Thiscell division is accomplished by the centromeres (poles) within a celldividing and form at each end of the cell. They go through a processthat separates the chromatin mass into equal parts, forming a new cell,and so on as new cells are formed. These centromeres act as positive andnegative poles, separating the chromatin mass equally at the two ends.In the case of the spermatozoa, the second metaphase shows the twospermatids, one being positive and the other negative. Thus, we have twospermatids carrying a positive (+) charge and two carrying a negative(-) charge. Two are X-spermatids and two are Y-spermatids, and uponmaturation, will be mature X- and Y-spermatozoa.

In the case of the egg (ova) the Y-polar body is thrown off and nevermatures or develops. Only the X-polar body of one is retained with theegg. Thus, the egg always carries the X-chromatin material.

During fertilization, this electro-potential energy of the sperm isneutralized due to literally hundreds of sperm, both X- and Y-, whichbombard the egg in an attempt to unite with it in the fertilizationprocess. This sets up a chemical neutralizing reaction around the egg,allowing only one of the spermatozoa to ultimately unite with the ova,regardless of the electro-potential of the spermatozoa. Without thisbombardment and chemical reaction around the egg, no fertilization wouldtake place. The present invention utilizes this electro-potential energyof the spermatozoa in the separation process of X- and Y-mammalianspermatozoa.

Spermatozoa of the X- and Y-types with different electro-potentialenergy are attracted to their opposite charge. In the case of theX-types of spermatozoa, they are negative (-) in polarity, therefore,they will migrate toward the positive (+) pole, whereas, theY-spermatozoa are of positive polarity and will migrate with the currentflow opposite to their charge. The passage of direct current through thecolumn in the present invention enhances the movement of the spermatozoain a directional force corresponding to the spermatozoa's oppositeelectro-potential energy. This electro-directional migration ofspermatozoa is true for an electrostatic field. Once the spermatozoa areout of the electrostatic influence, they revert back to their originalcharge.

It needs to be stated that in the case of some animals within a givenspecies, their spermatozoa separate more distinctly into twopopulations, using one or the other electro-forces. Our explanation forthis lies in the fact that the pH of the spermatozoa has an influenceupon the positive and negative ions incorporated within the semen sampleas well as the spermatozoa themselves. This is an important aspect as towhy these electro-forces influence the separation of the X- andY-spermatozoa in the present invention. It should be noted that pH isthe measurement of the H⁺ ion concentration that causes current to flowwithin an electrolyte system.

The respiratory column that removes the oxygen from the system aspreviously stated, is shown in FIG. 1 of the drawings of the apparatus.It is a closed system that utilizes manometric negative pressures forchanging the osmotic pressures within the system.

The addition of direct current force influence to the apparatus of FIG.1, is shown in FIG. 2.

The incorporation of the electrostatic force to the respiratory columnin FIG. 1, is shown in FIG. 3 of the drawings. FIGS. 4 and 5 arecontinuous flow systems which utilize the above principles.

The present invention for the separation of viable X- and Y-spermatozoautilizes commercial extenders, to extend the sperm samples used in theseparation process. These extenders are commonly used in artificialbreeding of animals within a species. This allows for a practical methodto handle semen samples as well as separation of the X- andY-spermatozoa.

Samples from the top and bottom of the apparatus, containing the samenumber of spermatozoa are subjected to measurements of how much oxygeneach population consumes. It has been found that one population X- orY-spermatozoa consumes oxygen at a different rate and amount over agiven time measurement.

One population of spermatozoa is acidophilic (acid-loving, spermatozoa)and the other population is basophilic (base-loving, spermatozoa).Samples from each population are subjected to acid and basic solutionsto determine the types of spermatozoa they contain.

Populations from each sample taken from the top and bottom of thepresent invention are subjected to a system that will measure particlesizes from 0.5 micron and larger. It will measure their diameter, lengthand/or width and will count the total number on the screen. In thismanner, slight variations in the size of the spermatozoa may bedetected. The X-spermatozoa is slightly larger than the Y-spermatozoa.

The foregoing is considered as illustrative only of the principles ofthe invention. Further, since numerous modifications and changes willreadily occur to those skilled in the art, it is not desired to limitthe invention to the exact construction and operation shown anddescribed, and accordingly, all suitable modifications and equivalentsmay be resorted to, falling within the scope of the invention.

What is claimed as new is as follows:
 1. A process for separating andcollecting viable female spermatozoa (XX chromosome) and malespermatozoa (XY chromosome) in a semen sample, which process comprisessubjecting the semen sample to negative pressure to cause the malespermatozoa to move to the top of the sample, wherein the semen sampleis brought into contact with a semen extender material, the semenextender material being disposed in surmounting relation to the sample,the male spermatozoa migrating into the semen extender material onsubjection of the sample and the semen extender material to negativepressure.
 2. The process of claim 1 and further comprising the step ofsegregating an upper portion of the sample, which upper portion containsa greater concentration of male spermatozoa, from a lower portion of thesample, which lower portion contains a greater concentration of femalespermatozoa.
 3. The process of claim 1 wherein the negative pressure isequivalent to a manometric pressure of 30 inches of mercury.
 4. Theprocess of claim 3 wherein the negative pressure is applied to thesample for approximately one hour.
 5. The process of claim 1 and furthercomprising the step of segregating the semen extender material from thesemen sample, which semen extender material contains a greaterconcentration of male spermatozoa.
 6. The process of claim 1 wherein thesample is subjected to a negative pressure different from the negativepressure applied to the semen extender material.
 7. The process of claim1 and further comprising the step of applying a direct electricalcurrent to the sample to increase separation of the female spermatozoafrom the male spermatozoa.
 8. The process of claim 7 wherein the directelectrical current is applied at a varying voltage of approximately sixvolts.